Florfenicol (FFC) is widely used to treat bacterial infections in veterinary medicine; however, its pharmacokinetic characteristics in reptiles remain limited. This study investigated the pharmacokinetic profiles of FFC after intramuscular (IM) injection at doses of 20 or 30 mg/kg body weight (b.w.) in freshwater crocodiles (Crocodylus siamensis). A sample of 10 healthy crocodiles was randomly divided into two groups (n = 5 for each group) according to a parallel study design. Blood samples were obtained from pre-dose to 168 h post-administration. Plasma FFC concentrations were quantified using high-performance liquid chromatography with diode array detection (HPLC-DAD) and analyzed by non-compartmental analysis. The mean maximum plasma concentrations of FFC were 4.05 µg/mL and 6.11 µg/mL for the 20 and 30 mg/kg b.w. doses, respectively. The mean elimination half-lives of FFC were long but not significantly different (51 h). The average plasma protein binding was 37.15%. Based on the pharmacokinetics/pharmacodynamics (PK/PD) index, a single dose of FFC via IM elicited plasma concentrations above the MIC90 values reported for several susceptible bacterial pathogens. Consequently, both dose levels provided plasma exposure consistent with previously reported reference MIC values. However, further PK/PD and multiple-dose investigations are needed to refine species-specific dosage regimens.