We studied the antimicrobial effect of gold quantum dots (AuQDs), femtosecond laser irradiation, and the combined effect of laser irradiation and AuQD treatment against common infectious eye pathogens. The INSPIRE HF100 laser system (Spectra Physics) provided a femtosecond laser, which was pumped by a mode-locked femtosecond Ti: sapphire laser MAI TAI HP (Spectra Physics), while a Quanta-Ray nanosecond Nd: YAG laser (Spectra-Physics) was used to precisely synthesize 7.8, 8.7, and 11.6 nm spherical AuQDs. Then, the in vitro growth kinetics and growth rate analysis of E. coli, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes, and Candida albicans (treated with the AuQDs, femtosecond laser irradiation, or combined laser and AuQDs treatment) was measured. The biocompatibility of the AuQDs with the retinal epithelial cell lines (ARPE-19) and their toxicity to the cells was assayed. The results showed that (1) in vitro irradiation using a 159 J/cm2 energy density obtained from the 400 nm femtosecond laser suppressed the growth of each of the five pathogens. (2) Similarly, treatment with the AuQDs was antimicrobial against the four bacteria. The AuQDs with an average size of 7.8 nm were more highly antimicrobial and biocompatible and were less cytotoxic than the larger AuQD sizes. (3) The combined femtosecond laser irradiation and AuQD treatment was more highly antimicrobial than each treatment alone. (4) The AuQD treatment did not impair the rate of wound closure in vitro. These findings suggest that combined femtosecond laser irradiation and AuQD treatment is significantly antimicrobial against Candida albicans, Gram-positive L. monocytogenes, S. aureus, and E. faecalis, as well as Gram-negative E. coli. The nontoxicity and biocompatibility of the AuQD particles tested suggest that this form of treatment may be clinically viable.