Water Framework Directive requires reliable and effective monitoring tools, andhydroacoustics has a potential to be one of them. The effect of pulse duration onin situ acoustical estimates of fish density and their sizedistribution was investigated. Measurements were performed in the oligo-mesotrophic LakeHancza (Poland) using a SIMRAD EK60 split-beam echo-sounder at 70 kHz frequency. Duringthe survey, two similar transducers pinged alternatively through the multiplexer using 4different pulse lengths, from short to long ones. The results show that the volumebackscattering coefficient (Sv) values, equivalent of the fish biomass, were notinfluenced by the pulse length. However, the number of the detected fish, the mean targetstrength (TS), and consequently the fish density, differed significantly for the longpulse duration data. This was especially noticeable in the layer above the thermoclinewith dense fish populations. In this upper layer, for the long pulse the Sawada indexfrequently exceeded value of 0.1 leading to overestimation of the mean TS andunderestimation of the fish density.