The efficacy of various lithium chloride (LiCl) applications in eradicating the parasitic mite Varroa destructor in honey bee colonies was investigated, with a specific focus on its impact on brood development. In broodless colonies (3 weeks post queen caging), the highest efficacy of 98% was achieved with a 9-day treatment of 2.5 kg of candy spiked with 50 mM LiCl. A shorter 5-day treatment with 2 kg of 50 mM LiCl candy resulted in an efficacy of 78%. In colonies with brood, a repeated short-term application of 4 × 0.5 kg 50 mM LiCl candy yielded an efficacy of 88%. LiCl treatment led to a removal of the first batch of brood reared after release of the queen. However, no long-term effects on colony growth were observed, and the colonies successfully overwintered. Additionally, the study demonstrated that lithium is rapidly distributed among the bees of a colony within 2 days, yet only low concentrations were detected in stored food samples. This suggests that the bees efficiently absorb and distribute lithium within the colony. The harvested honey in the following spring revealed a lithium concentration of 0.1–0.2 mg/kg, which is below naturally occurring lithium levels in honey. Based on these findings, LiCl can be considered an effective and easy-to-apply acaricide in broodless colonies, and even in colonies with brood, it had good efficacy and no long-term effects on colony survival. Further research may be necessary to determine the optimal treatment period for achieving an efficacy over 95%.