Urinary tract infection (UTI) is a very common infection in humans and a majority is caused by Escherichia coli. UTI are commonly treated empirically. However, empiric treatment has become more problematic due to increased antibiotic resistance to commonly used antibiotic agents. It is therefore desirable with short turnover times for antimicrobial susceptibility testing and species identification to improve antibiotic treatment at an early stage. Matrix Assisted Laser Desorption/Ionisation Time of Flight (MALDI-TOF) can provide species identification faster than former routine methods. This study compared direct and standard susceptibility testing using disk diffusion on Enterobacteriaceae (EB) from urine samples. The possibility to standardise the inoculum for direct susceptibility testing via a pellet obtained by a series of centrifugations was also evaluated, as well as direct species identification with MALDI-TOF from the pellet. Results from direct susceptibility testing from urine samples with EB, performed either directly from the urine or with a standardised inoculum, correlated well to those obtained with standardised susceptibility testing using EUCAST disk diffusion methodology with few errors, of which most were associated with Proteus mirabilis. The concept of standardising the inoculum for direct susceptibility testing to 0.5 McFarland was labour intensive and did not improve the results further. However, direct species identification from the urine pellet using MALDI-TOF showed good correlation to routine identification. Of 238 samples, an EB was correctly identified in 148 samples using MALDI-TOF.