FAME is the most common renewable component of conventional automotive diesel fuel offering environmental benefits when it is used in internal combustion engines. Despite the advantages, biodiesel is more susceptible to oxidative deterioration and due to its chemical composition as well as its high affinity to water, is considered to be a favorable substrate for microorganisms. On the other hand, apart from biodiesel, alcohols are considered to be promising substitutes to conventional diesel fuel because they could offer higher oxygen concentration leading to better combustion characteristics and lower exhaust emissions. In specific, according to the literature, n-butanol is a renewable alcohol demonstrating better blending capabilities and properties when it is added in diesel fuel, as its composition is closer to conventional fuel, compared to ethanol in example. Taking into consideration the alleged disinfectant properties of alcohols, it would be interesting to examine also the microbial stability of blends containing n-butanol in various concentrations. Based on the aforementioned, the aim of this study is to investigate the effect of n-butanol in diesel/ biodiesel blends on fuel quality characteristics (ignition quality, lubricity) while the oxidation and microbial stability is also assessed. Blends of automotive diesel with a commercial FAME up to 20% v/v and n-butanol at various concentrations were prepared. Initially, the blending stability of the prepared samples was evaluated. Ignition quality of the blends was assessed according to the ASTM D7170:2014/ EN 16144 test method. The lubricity of the blends was determined according to EN ISO 12156-1 and oxidation stability measurements were carried out under accelerated oxidative conditions by ASTM D7545. Microbial stability of diesel/biodiesel/n-butanol blends was assessed and compared to diesel-biodiesel ones by preparing laboratory-scale contaminated microcosms, which were stored for a certain period of time. The microbial proliferation was monitored by employing standard testing protocols for detecting microbiological activity in the fuel supply chain. At the end of storage time, the quality parameters of the blends were evaluated in order to determine any alterations.