Derby was forced to play with only under-23 and under-18 players after its first-team squad and interim manager Wayne Rooney were ruled out after a virus outbreak at the Championship club.
Chorley, from the sixth-tier National League North, took advantage of Derby's weakened line-up, with all its players making their debuts and with an average age of 19, reaching the fourth round for the first time.
It was Chorley's third league scalp in the FA Cup this season after previous wins against Wigan Athletic and Peterborough United.
In freezing conditions, with snow cleared off the pitch before kick-off, Chorley went ahead after 10 minutes when Derby failed to clear a long throw and Connor Hall nodded over the line.
Chorley's modest Victory Park ground is so homely that one fan was able to watch from over a back-garden fence, while two others used a cherry picker crane for a glimpse of the action.
Those enterprising fans saw Chorley's Mike Calveley seal their side's memorable success when he poked home from close range in the 84th minute.
The cup upset was a welcome boost for Chorley manager Jamie Vermiglio, whose job as a primary school headteacher has been thrown into chaos by the current national lockdown in Britain.
"It's a very proud moment for us. Credit to our boys, they've been on a journey and we said it before the game, the journey can continue today and it has done," Vermiglio said.
There are growing concerns over whether the English season should be halted as the new COVID-19 variant wreaks havoc across the country.
Liverpool beat virus-plagued Aston Villa 4-1 on Saturday (AEDT), with its opponents missing the entire first-team squad and boss Dean Smith because of a COVID outbreak.
Southampton's third-round tie against Shrewsbury on Monday (AEDT) has been postponed after several positive tests at the League One team.
A record-high 40 positive tests were detected among top-flight players and staff in the week between 28 December and 3 January and four Premier League games have already been postponed this term.
Newcastle United boss Steve Bruce and Leeds United manager Marcelo Bielsa claim there is a moral argument to suspend all matches with the UK death toll from the virus reaching a new high this week with 1325 deaths reported on Saturday (AEDT) alone.