The purpose of this study was to prepare orange oil microemulsion (ME) and to investigate the antimicrobial activity of film containing orange oil ME. First, surfactants and co-surfactants were screened on their efficiency to form ME using pseudo-ternary phase diagrams. The influences of surfactant and co-surfactant mass ratios were studied and optimized ME-loaded-films were prepared. Then, films containing orange oil ME were characterized by SEM and texture analyzer, and then evaluated for antimicrobial activity against Staphylococcus aureus and Propionibacterium acnes using an agar disc diffusion method. The results showed that Tween 80 as surfactant and propylene glycol as co-surfactant at a 1:1 ratio possessed the maximum ME area. Three ME formulations of ME 20, ME 25, and ME 30, which consisted of 20, 25, and 30% w/v of orange oil were prepared, respectively. All ME formulations showed particle sizes of about 60.26–80.00 nm, with broad a polydispersity index of 0.42. The orange oil ME films exhibited higher elastic values than the control. The diameters of inhibition zones for FME 20, FME 25, and FME 30 against P. acnes were 13.64, 15.18, and 16.10 mm, respectively. Only the FME 30 had an antimicrobial activity against S. aureus with 8.32 mm of inhibition zone. Contrarily, the control film had no antimicrobial activity against both bacteria. In conclusion, the present study found that the antibacterial activity of orange oil in pectin thin film could be enhanced by preparing orange oil as an ME before loading into pectin thin film.