In vitro studies were conducted to assess the sensitivity of Streptococcus iniae, the etiological agent of streptococcusis of rainbow trout (Oncorhynchus mykiss) to the antibacterial drugs florfenicol (FFC) and nano florfenicol (NFFC). The isolates originated from rainbow trout (Oncorhynchus mykiss) infected with Streptococcus iniae through natural outbreaks of streptococcusis. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) and Kirby- Bauer zones of inhibition (in mm) for FFC and NFFC against S. iniae were determined. The results showed that the MIC for NFFC and FFC tested with S. iniae were 0.078µg/ml and 0.312µg/ml respectively. The MBC for NFFC were 0.156μg/ml and for FFC 0.312μg/ml. There were statistically significant difference in the MIC and the MBC between NFFC and FFC (p<0.05). The diameter of inhibition zone for NFFC and FFC were 32.80±1.1mm and 31.63±0.4 mm respectively that there was no significant differences statistically (p>0.05). It is concluded that these drugs, especially nano-florfenicol, have a high potential for controlling Streptococcus iniae. Therefore, nano-florfenicol can be considered as the basis for antibiotic-resistant bacteria.