The two sides will lock horns in the competition for the second successive season, with Nick Cushing's side looking to avenge last year's Round of 32 defeat.
The pair also met in pre-season at the Women's International Champions Cup, as Tessa Wullaert struck in injury time to clinch a superb comeback victory.
City will host the Spanish outfit at the Academy Stadium in the first leg on Wednesday 16 October at 7pm BST, before travelling to Madrid on Wednesday 30 October at 7:30pm CET.
Cushing's side reached the semi-finals of the competition in both 2017 and 2018, before being eliminated by eventual Champions Lyon on both occasions. Our third European campaign was halted at the first hurdle with a 3-1 aggregate defeat to Atletico, following a 1-1 draw in the away leg and a 2-0 home defeat.
City reached this stage of the 2019/20 edition with an 11-1 aggregate triumph over Lugano, courtesy of a 7-1 victory in the away leg and 4-0 home success. Madrid defeated Spartak Subotica 4-3 over two legs, triumphing 3-2 in Serbia.
October's clashes will also see former City star Toni Duggan face her former Club.
Glasgow City v Brondby IF
Barcelona v FC Minsk
BIIK-Kazygurt v Bayern Munich
Lyon v Fortuna Hjorring
Paris Saint-Germain v Breidablik
Wolfsburg v FC Twente
Slavia Praha v Arsenal
City v Atletico Madrid
The quarter and semi-final draws will be conducted on Friday 8 November, 2019 before the competition then takes a break, resuming in March for the quarter-finals on 24 or 25 March (first leg) and 1 or 2 April.
The semi-finals will be played on 25 and 26 April (first leg) and 2 or 3 May 2019.
The final will be held at Viola Park in Vienna, Austria on Saturday 24 May 2019.