Abstract: This study aimed to assess antimicrobial susceptibility of members of the family Flavobacteriaceae isolated from Nile tilapia (Oreochromis niloticus). Antimicrobial susceptibility of 67 Flavobacteriaceae isolates originating mainly from ponds and Lake Victoria against 19 antimicrobial agents was determined by the broth micro dilution method. Overall, most isolates were susceptible to enrofloxacin (97%; MIC90 2μg/ml) followed by novobiocin (85%, MIC90, 4μg/ml) and the aminoglycoside streptomycin (85%; MIC90, 128μg/ml). Some isolates were also susceptible towards trimethoprim/sulfamethoxazole (77.6%), neomycin and florfenicol both at 62.7%. Susceptibility levels were low for tylosin tartrate (32.8%), clindamycin and sulphathiazole both at (23.9%), ceftiofur (6%), spectinomycin (6%) and tetracyclines/ oxtetracyclines (4.5%). In contrast, β-Lactams (amoxicillin, penicillin), gentamycin and erythromycin exhibited very poor activity against Flavobacteriaceae isolates. The extent of antimicrobial susceptibility did not vary significantly among isolates from farmed and wild fish isolates (P > 0.01). The highest Multiple Antimicrobial Resistance (MAR) index was observed in Chryseobacterium indologenes (0.89) and the lowest in C. indicum isolates (0.32). Our results indicate that most of Flavobacteriaceae isolates are multidrug resistance, and this may be associated with intrinsic resistance mechanisms to a broad range of antimicrobial agents. However, the need remains to carryout in-depth study to understand better the underlying genetic mechanisms given that the magnitude and trend for susceptibility was comparable between isolates from aquaculture and fisheries. The findings from this study give us insight into appropriate choice of antimicrobial agents for effective treatment of infections caused by these isolates.