Nowadays, sustainable biofuel production is a hot topic. However, studying the processing of new alternative materials on an industrial scale is very expensive. According to this fact, testing on pilot plants is a suitable method for studying the effects of alternative feedstocks on catalyst activity or product quality parameters. The present work deals with the effect of coprocessing used cooking oil (5−30 wt %) with refinery petroleum fractions on product properties and hydrotreating catalyst activity. The experiment was performed on a pilot unit at industrial operating conditions (5.5 MPa, WHSV 1.1 h −1 , H 2 /feed ratio 327 Nl•l −1 ) using a commercial NiMo/γ-Al 2 O 3 catalyst. Operating temperature (341−352 °C) played the most significant role in catalyst activity to get products with 10 mg•kg −1 sulfur content. The obtained products were evaluated based on the standard analytical methods specified in the EN 590 standard. Furthermore, the following advanced analytical methods were chosen for qualitative and quantitative analyses: GCxGC-FID, GCxGC-SCD, Fourier transform infrared spectroscopy with attenuated total reflectance FTIR-ATR, and Raman spectroscopy. The increase of used cooking oil (UCO) on the feed during coprocessing increased nC 15 −nC 18 alkanes with the consequent changes in the product properties such as density at 15 °C and cetane index. The increase in light gases (C 1 −C 4 ) and CO 2 indicates the promotion of the decarboxylation pathway during coprocessing. Overall, our results indicate the necessary changes in the operating conditions during coprocessing to get the EN 590 requirements with up to 30 wt % of UCO in the feed, which is the line of future advances for biofuel production at the industrial scale.