Essential oils have attracted growing attention in the medicine field due to the significant bioactivities. The purpose of present study was devoted to optimizing the microwave-assisted hydrodistillation (MAHD) of sugarcane molasses essential oils (SMEO), compared with chemical components and extraction efficiency of SMEO using MAHD and hydrodistillation (HD) methods, and evaluating antioxidant and antibacterial activities of SMEO extracted by MAHD and HD methods. In this study, microwave-assisted hydrodistillation (MAHD) was first developed to extract SMEO, and MAHD was optimized through single-factor test and response surface methodology (RSM). The optimal processing conditions of SMEO were 60 min of extraction time, 500 W of power, and 1.95 mL/mL of ratio of liquid to solid, and the yield of SMEO was 0.0788 +/- 0.002 \% (v/v). Compared with yield of SMEO, The MAHD method was more efficient than hydrodistillation (HD) method. Moreover, chemical compositions of SMEO using MAHD and HD methods were identified and compared by gas chromatography-mass spectrometry (GC-MS). Principal chemical components were found in SMEO using MAHD and HD methods, but it was in different content. The antioxidant activity of SMEO was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, and SMEO using MAHD method showed a better antioxidant activity than SMEO using HD method. The antibacterial activity of SMEO were evaluated by determining the diameters of inhibition, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC), and the result exhibited that the antibacterial activity of SMEO using MAHD method was higher than SMEO using HD method. The results of scanning electron microscopy (SEM) exhibited that the SMEO using MAHD method changed microstructure of Escherichia coll. and Sthaphylococcus aureus. Therefore, the MAHD technology significantly improved the yield of SMEO, and the biological activity results showed a promising alternative and provided a foundation for application of SMEO obtained by MAHD method in food, pharmaceutical, and sanitizers industries.