Non-lethal biopsy methods (including blood, gill, and muscle biopsies) have been used to study the health and physiological status of wild fishes. Nonetheless, concerns exist regarding the impact of non-lethal sampling on relevant welfare measures such as behaviour and survival. Here, nesting Smallmouth Bass (Micropterus dolomieu) were used as a model species to study in situ how fish respond to non-lethal sampling. Male Smallmouth Bass provide sole parental care and guard well-defined nests for a period of several weeks, providing a unique opportunity to assess behaviour, reproductive success, and survival in the wild. Fish were captured from their nests by angling and subjected to a biopsy (either blood, gill, or muscle), or a combination of all three biopsy methods prior to release. A control group that was captured but not biopsied as well as a non-angled control were also included. Nests were monitored for a period of four weeks or until the parental males either abandoned offspring, died, or raised a brood to independence. Single biopsies, regardless of the biopsy type, were found to have no impact on parental care and survival, but fish that received the combined treatment took longer to return to their nest and displayed a 6.5 times greater likelihood of nest abandonment. Mortality was only observed in fish that received the combined biopsy treatment. As such, this study reveals that it is possible to maintain the welfare status of Smallmouth Bass in the wild by using individual biopsies, thus emphasizing the importance of making careful decisions about which tissues are needed to achieve desired study objectives. This is one of the few studies to assess the behavioural and fitness consequences of increasingly common non-lethal biopsy methods and provides useful information on the relative consequences of different biopsy methods on wild fish.