Pacific lamprey Entosphenus tridentatus are facing severe population declines, yet little is known about juvenile lamprey passage, life history, or adult return rates because until now, these small fish could not be tagged for unique identification of live individuals. Previously, we developed a simple and effective method for tagging juvenile lampreys with passive integrated transponder (PIT) tags and showed that tagging per se did not affect survival. Mortality in tagged and untagged control fish, however, was frequently associated with fungal infection. In this study, we addressed two outstanding issues related to handling and tagging juvenile lampreys. First, we tried to mitigate freshwater fungal infections by reducing irritation and stress from anesthesia and by treating tagged fish briefly with a prophylactic immediately after tagging. We tested four anesthetics at three concentrations each and determined that 100 mg/L MS-222 and 60 mg/L BENZOAK^® (benzocaine) were the most effective for anesthetizing juvenile lampreys to handleable while minimizing irritation. We also showed that fish anesthetized with BENZOAK^® may have lower rates of fungal infection than those anesthetized with MS-222 or AQUI-S^® 20E (eugenol). When fish anesthetized with MS-222 or BENZOAK^® were given a 30 min prophylactic treatment with Stress Coat^®, hydrogen peroxide, or salt immediately after tagging, few fish presented with fungal infections. However, untreated, tagged control fish also showed few fungal infections, making it difficult to determine if the prophylactic treatments were successful. The second question we addressed was whether activity would increase tag loss in PIT-tagged lampreys. We found that active swimming did not cause tag loss if fish were first held for 20–24 h after tagging. Therefore, we recommend anesthesia with MS-222 or BENZOAK^® and then tagging with a 20–24 h recovery period followed by immediate release. If field studies show that lampreys are not reaching salt water (where fungal infections are mitigated) within 1–2 weeks after release, further study of prophylactic treatments may be warranted.