The Bedfordshire side hailed the win as their best of the season, but the Titans battled well and were unlucky not to take something from the game.
Centre Ellis Abrahams’s penalty on 33 minutes was their only score of a first half which ended 7-3, and despite playing uphill after the break Rotherham took the lead with centre Jamie Cooke touching down out wide on 42 minutes.
The two sides remained matched until the final 17 minutes, with Ampthill scoring two tries, a conversion and penalty to seal the win.
Hull Ionians stretched their lead in National Two North after they beat Huddersfield 20-15 in a clash of the top two.
Huddersfield started brightly and went in front after 15 minutes, but it was the Ionians pack that made the impact with all three of their tries coming from the forwards, Ed Falkingham with the last to see them over the line.
Despite four second-half tries and five in total from Leicester Lions, Wharfedale took home the spoils as they moved up to sixth with a 34-30 home win.
The first half was dominated by fly-half Jack Blakeney-Edwards with all 13 points, and the Yorkshire side made an equally quick start to the second, running over the line twice through Philip Woodhead and Rhys Lovegrove.
Three tries brought Leicester back to within two points, but the hosts were awarded a penalty try nine minutes before time to seal the win.
Sheffield Tigers had a day to remember as they ran in eight tries to record a 54-7 victory over strugglers Macclesfield.
Four tries before the half ended helped Tigers race into a 28-0 lead at the end of the first half. The second half was much the same but it would be the hosts’ outside centre Jamie Broadley that stole the show, adding three more tries to his one from the first half.
Elsewhere, Otley crossed the line four times to secure a bonus point 28-10 win over Peterborough.
Leaders Hull maintained their recent good form with a deserved 23-8 win at Yorkshire rivals Ilkley in the North Premier league.
The visitors crossed the line three times and the defence –which has conceded the fewest points in the league – were at their ruthless best to hold their opponents to registering single figures.
Vale of Lune and Harrogate battled out the game of the day, with the Yorkshire side securing a 45-38 win in a 12-try thriller in Lancashire.
Sandal narrowly missed out on victory and a top four spot as their trip to Kirkby Lonsdale ended with a 19-18 defeat.
It’s close at the top of the North One East table with four teams level on 33 points.
York dropped to second place as they were beaten 34-24 at Huddersfield YMCA who move up to third, while Scarborough also lost, 42-26 at home against Driffield, as they drop to fourth in the standings.
Elsewhere, Malton and Norton secured a much-needed 33-29 win over Morley, while West Leeds went down 34-9 to new leaders Morpeth.
Bradford and Bingley ran in five tries on their way to a comfortable 54-22 win over Consett, and Cleckheaton were narrow 20-17 winners over Pocklington.
Leaders Old Crossleyans were deserved 26-5 winners at rivals Old Rishworthians in Yorkshire One, while second-placed Moortown kept pace with them following an impressive 48-15 win over Middlesbrough.
Heath’s 31-19 win at Guisborough leaves them in third position and just four points away from top spot, while Old Brodleians were also able to keep pace with the top sides after a 40-27 win over Selby moved them up to fourt in the standings.