This research aimed to obtain biocompatible and antimicrobial nanofibres based on concentrated collagen hydrolysate loaded with thyme or oregano essential oils as a natural alternative to synthesis products. The essential oils were successfully incorporated using electrospinning process into collagen resulting nanofibres with diameter from 471 nm to 580 nm and porous structure. The presence of essential oils in collagen nanofibre mats was confirmed by Attenuated Total Reflectance -Fourier Transform Infrared Spectroscopy (ATR-FTIR), Ultraviolet–visible spectroscopy (UV–VIS) and antimicrobial activity. Scanning Electron Microscopy with Energy Dispersive Spectroscopy analyses allowed evaluating the morphology and constituent elements of the nanofibre networks. Microbiological tests performed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans showed that the presence of essential oils supplemented the new collagen nanofibres with antimicrobial properties. The biocompatibility of collagen and collagen with essential oils was assessed by in vitro cultivation with NCTC clone 929 of fibroblastic cells and cell viability measurement. The results showed that the collagen and thyme or oregano oil composites have no cytotoxicity up to concentrations of 1000 μg·mL−1 and 500 μg mL−1, respectively. Optimization of electrospinning parameters has led to the obtaining of new collagen electrospun nanofibre mats loaded with essential oils with potential use for wound dressings, tissue engineering or protective clothing.