Cigarette butts (CB) comprise the most abundant form of litter worldwide, as an estimated 4.5 trillion cigarette butts are littered into the environment each year. Since the 1980s, cigarette butts have consistently comprised 30 to 40% of all items collected in annual international coastal and urban cleanups. Under normal conditions, cigarette butt filters may take up to 18 months to biodegrade. Due to surface erosion and rainfall, cigarette butts can travel downstream and deposit into our oceans and inland waterways. Furthermore, cigarette butts can leach toxic chemicals trapped in their filters after exposure to a water source. Previous studies have demonstrated the ability of smoked cigarette leachate to exert acute and chronic toxicity to a variety of aquatic organisms. However, the bioaccumulative capacity of these tobacco contaminants in aquatic organisms exposed to cigarette butt leachate as well as human exposures through the food chain remain unknown. Using standard fish bioassays, the bioaccumulation potential of organic compounds from smoked cigarette litter was assessed in the freshwater rainbow trout (Oncorhynchus mykiss) following direct exposure to 0.5 CB/L leachate for a 28-day period. A non-targeted analysis was conducted to identify chemical constituents accumulated in the fish. Notably, four organic tobacco alkaloids, nicotine, nicotyrine, myosmine, and 2,2’-bipyridine, were identified in fish directly exposed to smoked cigarette litter leachate. An average tissue concentration of 466 ng/g, 55.4 ng/g, 94.1 ng/g, and 70.8 ng/g was found for nicotine, nicotyrine, myosmine, and 2,2’-bipyridine, respectively. Bioconcentration factors of 0.270, 84.2, 14.4, and 532 were calculated for nicotine, nicotyrine, myosmine, and 2,2’-bipyridine, respectively. A significant reduction in fish weight was observed between the lab control (0.0 cb/L) and 0.5 cb/L exposures (Nested ANOVA, F1,4 = 10.2, p = 0.0329; medium to large effect size of 0.72). In contrast, no significant variation in fish weight was found among tanks within each exposure (Nested ANOVA, F4,79 = 0.955, p = 0.437). The data presented in this study is the first to suggest the bioaccumulative capacity of leachable organic compounds from smoked cigarette litter and their latent ability to disseminate throughout the food chain, ultimately resulting in the potential for human exposure.