The aim of this research was to analyze the relationship among hygienic behavior (HB), Varroa destructor infestation, and honey production in the central highlands of Ecuador. Overall, 75 honey bee colonies were evaluated before, during, and after production at three altitude levels (2600–2800, 2801–3000, and >3000 m.a.s.l.). The hygienic behavior percentage of the colonies was determined by the pin-killing method, and the colonies were classified into three groups: high HB (>85%), mid HB (60.1–85%), and low HB (≤60%). Varroa infestation was diagnosed as well, and honey production was evaluated only during production. HB was high and heterogeneous, averaging 80% ± 9.7%. Its highest expression was observed at lower altitudes. The infestation degree was low (3.47% ± 1.56%), although the mite was detected in all colonies upon sampling. A negative correlation was observed between HB and Varroa infestation in the first sampling (−0.49 **), suggesting that the high- and mid-altitude HB colonies underwent the lowest infestation rates, regardless of sampling. The correlations between HB and production were significant (0.26 *), indicating a positive effect of HB on production, meaning that colonies with high HB obtained the highest honey production (25.08 ± 4.82 kg/hive). The HB of bees showed an inverse relationship with altitude and it tended to reduce the effect of Varroa infestation, favoring honey production and, thus, suggesting the feasibility of selecting colonies with high HB.