Ocimum basilicum L., is most popular fragrant herb renowned for its immense application. However, a plethora of studies revealed terpenoids and phenylpropanoids as marker chemicals with known identities. We herein report two unique chemotypes developed in CSIR-CIMAP for their compositional variability and biological activities. Essential oils obtained from aerial parts of both the chemotypes were analyzed in capillary gas chromatography and mass spectrometry systems on 5% diphenyl- and cyclodextrin coated fused silica columns. The statistical analysis was carried out to validate the phytochemical data of consecutive years and also to calculate the variability. The phenylpropanoids dominated the volatile fractions of chemotype II (72.5–77.5%) in comparison to chemotype I (29.8–40.0%) essential oil. In the contrary, sesquiterpenoids (16.4–17.4%) and monoterpenoids (33.5–43.9%) contributed oil composition of chemotype I in diverse proportions. The presence of single images of R-(+)-camphor and R-(-)-linalool were established in both the oils analyzed on a ethyl substituted cyclodextrin-based column. Essential oils were tested for biological activities against C. albicans, S. aureus, and E. coli. meta-Eugenol, the marker compound isolated from chemotype I showed best inhibition against C. albicans. In conclusion, meta-eugenol-rich chemotype I with high essential oil yield (0.1–0.8%) was identified as an alternative source for meta-eugenol.