The objective of this study was to evaluate physiological and biochemical alterations during the development and storage of habanero pepper seeds with a view toward determining the time of harvest. Seeds were manually extracted from the fruit at three stages of development: E1 (fruit with first signs of yellowing), E2 (mature fruit) and E3 (mature fruit submitted to seven days of rest). After drying, seeds with 8% water content were stored at 10°C for 0, 4 and 8 months, and their quality evaluated by means of germination and vigor tests. Activities of the enzymes α-amylase, endo-β-mannanase, esterase, Superoxide Dismutase (SOD), malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were evaluated during germination at 0, 48, 96 and 144 h after seeding. A randomized block design was used in a 3 × 3 factorial design (stages of development × storage) with 4 replications. Lower germination and vigor values were observed for the E1 stage seeds at all storage periods. In recently stored seeds, greater germination and vigor values were observed for the E3 stage seeds. Dormancy was observed principally in recently stored seeds and this was overcome at four months of storage. In summary, the physiological tests and activity of the enzymes evaluated indicated that the habanero pepper should be harvested at the E3 stage for a higher seed quality.