Chiral organic contaminants, like α-hexachlorocyclohexane (α-HCH), showed isotope fractionation and enantiomer fractionation during biodegradation. This study aims to understand the correlation between these two processes. Initial tests of α-HCH degradation by six Sphingobium strains (with different LinA variants) were conducted. Results showed variable enantiomer selectivity over the time course. In contrast, constant enantiomer selectivity was observed in experiments employing (i) cell suspensions, (ii) crude extracts, or (iii) LinA1 and LinA2 enzymes of strain B90A for α-HCH degradation in enzyme activity assay buffer. The average value of enantioselectivity (ES) were −0.45 ± 0.03 (cell suspensions), −0.60 ± 0.05 (crude extracts), and 1 (LinA1) or −1 (LinA2). The average carbon isotope enrichment factors (ε_c) of (+)α- and (−)α-HCH were increased from cells suspensions (−6.3 ± 0.1‰ and −2.3 ± 0.03‰) over crude extracts (−7.7 ± 0.4‰ and −3.4 ± 0.02‰) to purified enzymes (−11.1 ± 0.3‰ and −3.8 ± 0.2‰). The variability of ES and the ε_c were discussed based on the effect of mass transport and degradation rates. Our study demonstrates that enantiomer and isotope fractionation of α-HCH are two independent processes and both are affected by underlying reactions of individual enzymes and mass transport to a different extent.