Moxifloxacin is a fourth-generation fluoroquinolone antibiotic available for ophthalmic use. It inhibits two enzymes involved in bacterial DNA synthesis, covering Gram-positive and Gram-negative pathogens. This spectrum allows for the formulation of self-preserving bottle solutions, while its interesting pharmacological profile is distinguished by efficacy at low tissue concentrations and by an infrequent dose regimen due to its long duration on ocular tissues. This enhances patient compliance, promoting its use in children. The human eye hosts several microorganisms; this collection is called the ocular microbiota, which protects the ocular surface, assuring homeostasis. When choosing an antibiotic, it is appropriate to consider its influence on microbiota. A short dose regimen is preferred to minimize the impact of the drug. Moxifloxacin eyedrops represent an effective and safe tool to manage and prevent ocular infections. As healthcare providers face the complexity of the ocular microbiota and microbial resistance daily, the informed use of moxifloxacin is necessary to preserve its efficacy in the future. In this regard, it is well known that moxifloxacin has a lower capacity to induce resistance (an optimal WPC and MPC) compared to other quinolones, but much still needs to be explored regarding the impact that fluoroquinolones could have on the ocular microbiota.