Microorganisms can cause many operational problems, particularly, during storage and handling of fuel systems. The susceptibility of diesel systems to microbial contamination has been studied for many years but the introduction of biodiesel (Brazil-B5) has raised the incidence of problems in tanks around the world. Among the mitigation alternatives, biocides have been identified as a good one to curb microbial growth. The aim of this research was the effectiveness assessment of two biocides a MBO antimicrobial agent (as multifunctional package) and MIT/CMIT antimicrobial agent in biodiesel (B100 - 60% soya and 40% tallow), conventional diesel (B0 - low sulfur 50 ppm) and blends B7 and B10. The efficacy of two biocides was determined by evaluating the changes in a set of parameters during 60-days exposure to an uncharacterized microbial inoculum. Microcosms contained a fuel phase (B0, B100, B7 or B10) and two types of aqueous phase: natural bottom-water formed in B5 storage tank or a synthetic water with three levels of contamination: low (10(3) CFU L-1), medium (10(5) CFU mL(-1)) and high (10(8) CFU mL(-1)). Fuel phase as received and without biocide with sterile aqueous phase was used as a control and the sampling times were at 0, 7, 14, 21, 42 and 60 days. The fuel phase was rated by Haze scale (ASTMD 4176) and infrared analysis. Water phase parameters included: microbial viability (time-kill), presence of emulsion/biofilm and dry weight of biomass formed at fuel/water interface. The results suggest that antimicrobial MBO product as a multifunctional package can only control the microbial population when the microbial contamination level is low. The antimicrobial MIT/CMIT product was effective in all conditions (as received, media and high microbial contamination). (C) 2013 Elsevier Ltd. All rights reserved.