Twentieth Century Fox fired Singer as director of the project in December 2017 after rumours of clashes on the set with Malek and other cast members.
The "Bohemian Rhapsody" star had a seemingly uncomfortable when his famous friend appeared to snub him and turn her back on stage as he went to shake her hand while collecting the Best Picture prize.
Rami Malek, who won raves for his full-throated portrayal of the late Mercury, won best actor in a drama and thanked Mercury onstage "for giving me the thrill of a lifetime".
"What an honour. Thank you", he captioned the image after the Sunday night ceremony.
Bohemian Rhapsody broke box office records previous year, and has just won two Golden Globes Sunday's awards. It also bagged the biggest movie award of the night - best drama. "I've known her for many years, despite [what] it may seem from that video, so I thought I could easily go up and say hi but in a moment like that it was. very awkward".
Having said that, congratulations to Rami Malek.
"So the fact that I knew Nicole [was there], I was like, 'Oh, here's a bit of safety, '" he continued.
Rami Malek previously told Radio X that he didn't "want to let Freddie down".
The film also took home the Globe for best film drama, beating the likes of Black Panther, If Beale Street Could Talk, BlacKkKlansman and A Star is Born (2018).
The Golden Globe Awards' 76th edition were presented at the Beverly Hilton Hotel, Los Angeles, by Canadian actress Sandra Oh and American actor Andy Samberg.
Streaming service Netflix, which has upended Hollywood by spending billions on original programming, won five Golden Globes across movies and television, including best foreign language film "Roma".