Abstract: Antibiotic residue, a kind of biomass, is classified as hazardous waste. However, it is considered a good biomass resource because it contains rich organic matter and bacterial protein with a calorific value equivalent to that of low-rank coal. The hydrothermal method uses high-temperature liquid water as the reaction medium and reactant, which has the characteristics of high energy, fast reaction speed, large material flux, convenient feeding, and high product separation efficiency, especially avoiding the evaporation of high water content of aquatic substances. Although bio-oil obtained from the noncatalytic hydrothermal process has a high calorific value, it exhibits negative characteristics, such as high oxygen and nitrogen and high viscosity, which makes it unsuitable for use as a fuel. Therefore, catalysts are needed to improve the quality of bio-oil. This paper investigates the hydrothermal liquefaction of bacterial residues into bio-oil under a retention time of 30–240 min at 220–300 °C. Results show that the maximum yield of bio-oil is 28.01% at 260 °C for 135 min. Catalyzed by six kinds of catalysts (HCOOH, CH₃COOH, K₂CO₃, Na₂CO₃, NaOH, and KOH), the highest yield of bio-oil is achieved with Na₂CO₃ (36.06%) and NaOH (36.31%). The content of hydrocarbons and their derivatives in the produced bio-oil is found to be relatively low at varying amounts of Na₂CO₃ and NaOH catalysts. The mass fraction of nitrogen-containing compounds in the alkali-catalyzed and acid-catalyzed bio-oil is 41.16%–49.74% and 57.62%–59.32%, respectively, with the best nitrogen removal obtained at a mass dosage of 8%. In particular, the contents of nitrogen compounds in the bio-oil catalyzed by Na₂CO₃ and NaOH are 29.12% and 35.67%, respectively. The best removal effect of oxygen is achieved at a dosage of 10%. Specifically, bio-oil components produced by Na₂CO₃ and NaOH contains 32.12% and 29.02% oxygen-containing compounds, respectively. Moreover, the higher heating value (HHV) of bio-oil produced with these catalysts is the largest, with an HHV of 33.3220 and 34.7320 MJ·kg⁻¹ for Na₂CO₃ and NaOH, respectively.