Propenylbenzenes, including isosafrole, anethole, isoeugenol, and their derivatives, are natural compounds found in essential oils from various plants. Compounds of this group are important and valuable, and are used in the flavour and fragrance industries as well as the pharmaceutical and cosmetic industries. The aim of this study was to develop an efficient process for synthesising oxygenated derivatives of these compounds and evaluate their potential biological activities. In this paper, we propose a two-step chemo-enzymatic method. The first step involves the synthesis of corresponding diols1b–5bfrom propenylbenzenes1a–5avialipase catalysed epoxidation followed by epoxide hydrolysis. The second step involves the microbial oxidation of a diasteroisomeric mixture of diols1b–5bto yield the corresponding hydroxy ketones1c–4c, which in this study was performed on a preparative scale usingDietziasp. DSM44016,Rhodococcus erythropolisDSM44534,R. erythropolisPCM2150, andRhodococcus ruberPCM2166. Application of scaled-up processes allowed to obtain hydroxy ketones1-4cwith the following yield range 36–62.5%. The propenylbenzene derivatives thus obtained and the starting compounds were tested for various biological activities, including antimicrobial, antioxidant, haemolytic, and anticancer activities, and their impact on membrane fluidity. Fungistatic activity assay against selected strains ofCandida albicansresults in MIC50value varied from 37 to 124 μg/mL for compounds1a,3a–c,4a,b, and5a,b. The highest antiradical activity was shown by propenylbenzenes1-5awith a double bond in their structure with EC50value ranged from 19 to 31 μg/mL. Haemolytic activity assay showed no cytotoxicity of the tested compounds on human RBCs whereas, compounds2b–4band2c–4caffected the fluidity of the RBCs membrane. The tested compounds depending on their concentration showed different antiproliferative activity against HepG2, Caco-2, and MG63. The results indicate the potential utility of these compounds as fungistatics, antioxidants, and proliferation inhibitors of selected cell lines.