Flavobacterium covae is the predominant species responsible for columnaris disease in Channel Catfish, posing significant challenges to the aquaculture industry. Despite its importance, there is a lack of consistency in the culture media used for the optimal growth of F. covae, delaying in vitro and in vivo studies due to the bacterium’s fastidious nature. The objective of this study was to evaluate and compare the efficacy of various culture media to support the growth of F. covae during initial resuscitation, preparation of a stationary-phase inoculum, and throughout the bacterial growth curve. Initial resuscitation was conducted at 28 °C and evaluated at 24, 48, and 72 h using various agar and broth media, including Nutrient Agar, Tryptic Soy Agar, Tryptic Soy Agar with Blood, Brain Heart Infusion Agar and Broth, Reasoner’s 2A (R2A) Agar and Broth, and Shieh Broth. Consistent growth was observed with R2A (Agar and Broth) and Shieh Broth, observing small, yellow-pigmented colonies in agar and turbidity in broth after 48 h of incubation at 28 °C. Subsequent inoculation to obtain a stationary-phase inoculum and generate growth curves was conducted in R2A broth and Shieh broth at 28 °C for 48 h with constant agitation. A microplate reader was used to measure optical density (OD600) hourly. Bacterial growth was consistently observed in R2A broth across all replicates, whereas Shieh Broth showed either no growth or minimal growth, likely due to its sensitivity to pH and complex preparation requirements. The bacterial growth curve results reported a final population of 7.6 log CFU/ml for R2A and 6.3 log CFU/ml for Shieh. The viability of the bacterial stock and freshness of the media were also found to significantly influence outcomes. In conclusion, R2A could be a reliable option for cultivation of F. covae, being a promising candidate for standardization in laboratory protocols.