Ocimum spp. are reputed for their essential oils and aroma chemicals that are highly valued in food and flavor industries. This study was conducted to evaluate the diurnal effect on essential oil yield and composition of four Ocimum species, viz. O. basilicum L., O. gratissimum L., O. americanum L., and O. kilimandscharicum Guerke grown at foothills of Uttarakhand, India. Essential oil content and composition were determined and compared as a function of different harvesting times viz. morning (6.00 a.m.), noon (12.00 p.m.), and evening (6.00 p.m.). O. basilicum, O. americanum, and O. kilimandscharicum (camphor type) gave higher essential oil yield when harvested at noon, while in O. gratissimum, higher oil yield was obtained in the evening, followed by noon and morning harvesting. Oil yield in O. kilimandscharicum (eugenol/methyl chavicol) chemotype was found in the order of: morning > noon > evening. Similar to oil yield, significant qualitative and quantitative differences in essential oil composition of O. gratissimum and O. kilimandscharicum chemotypes were observed, while in O. basilicum chemotypes, little quantitative changes in compositions were found due to different harvesting times of the day. The results obtained could be of economic significance for determining the most favorable time for harvesting.