Background: To assess the in-vitro efficacy of delafloxacin, a new fourth generation fluoroquinolone, against Staphylococcus vitreous isolates from patients with clinically diagnosed endophthalmitis. This is the first investigation of delafloxacin in ocular tissues. Methods: Intravitreal isolates of culture-proven S. aureus and S. epidermidis were identified between 2014 and 2018. Minimum inhibitor concentrations (MIC) were determined using ETEST strips. The antibiotic susceptibilities were tested against a panel of drugs including glycopeptides such as vancomycin, as well as traditional and newer fluoroquinolones (levofloxacin, moxifloxacin, and delafloxacin). Results: Of 45 total isolates identified between 2014 and 2018, 13% (6) were methicillin-resistant S. aureus (MRSA), 9% (4) were methicillin-sensitive S. aureus (MSSA), 53% (24) were methicillin-resistant S. epidermidis (MRSE), and 24% (11) were methicillin-sensitive S. epidermidis (MSSE). Among the fluoroquinolones, resistance rates were 61% for levofloxacin, 50% for moxifloxacin, and 12% for delafloxacin. Inter-class comparisons between delafloxacin and the two other fluoroquinolones demonstrated higher Gram-positive susceptibility to delafloxacin (p < 0.01). MIC90 values were lowest for delafloxacin (1.0 μg/mL) compared to levofloxacin (8.0 μg/mL) and moxifloxacin (8.0 μg/mL). Vancomycin was 100% effective against all isolates with MIC90 value of 0.75 μg/mL. Conclusion: Compared to levofloxacin and moxifloxacin, the newer fluoroquinolone delafloxacin demonstrated the lowest MICs values and lowest rates of resistance for Gram-positive in-vitro S. epidermidis and S. aureus vitreous isolates.