Mycobacteriosis is a common bacterial infection in laboratory zebrafish caused by several different species and strains of Mycobacterium, including both rapid and slow growers. One control measure used to prevent mycobacterial spread within and between facilities is surface disinfection of eggs. Recent studies have highlighted the effectiveness of povidone-iodine (PVPI) on preventing propagation of Mycobacterium spp. found in zebrafish colonies. We evaluated the effect of disinfection using 12.5-50 ppm PVPI (unbuffered and buffered) on zebrafish exposed at 6 or 24 h postfertilization (hpf) to determine if this treatment is suitable for use in research zebrafish. Our results show that 6 hpf embryos are less sensitive to treatment as fewer effects on mortality, developmental delay, and deformity were observed. We also found that buffered PVPI treatment results in a greater knockdown of Mycobacterium chelonae and Mycobacterium marinum, as well as results in decreased harmful effects on embryos. Treatments of shorter (2 min vs. 5 min) duration were also more effective at killing mycobacteria in addition to resulting in fewer effects on embryo health. In addition, we compared the efficacy of a rinsing regimen to rinsing and disinfecting. Based on the findings of this study, we recommend disinfecting embryos for 2 min with buffered PVPI at 12.5-25 ppm.