Background: Moxifloxacin is a bactericidal methoxyquinolone used for the treatment of conjunctivitis and prophylactic therapy in cataract and refractive surgeries. Chloramphenicol is a bacteriostatic organochlorine introduced into clinical practice in 1948 and used mainly in topical preparations because of its known toxicity. Objectives: The study aimed to evaluate the in vitro antibacterial effect and the ocular cytotoxicity of these broad-spectrum antibiotics. Methods: Antimicrobic activity was tested on four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis), and determined through calculation of Minimum Inhibitory Concentration (MIC) and IC50 for each microorganism. Antibacterial activity was determined by microdilution method after 24 hours incubation with 2-fold serial dilutions (2.5 mg/ml to 4.883 µg/ml) of moxifloxacin and chloramphenicol. Disk diffusion test were performed according to EUCAST methodology. Biofilm formation inhibition (BIC) and biofilm Eradication Concentration (BEC) assay were conducted for P. aeruginosa and S. epidermidis using the microdilution method. Cytotoxicity of antibiotics was evaluated by MTT colorimetric assay on human corneal cell (HCE-2). Results: Cytotoxicity of antibiotics was evaluated on human epithelial corneal cells after 4 hours treatment by viability assay. Results showed that corneal cell viability was significantly higher after moxifloxacin treatment compared to Chloramphenicol (p<0.01). Moxifloxacin is characterized by a significantly lower MIC and IC50 values and a larger inhibition zone for all the strain tested, with high performance in controlling gram-negative growth, compared to Chloramphenicol. Moreover, Moxifloxacin showed higher activity compared to Chloramphenicol in the inhibition of biofilm formation and in the disruption of biofilm, especially against S. epidermidis biofilm. Conclusions: The lower corneal cell toxicity and the broader spectrum of antibacterial activity observed with Moxifloxacin suggests its use in ophthalmic solution for the treatment of bacterial eye infections.