Water Framework Directive requires reliable and effective monitoring tools, and hydroacoustics has a potential to be one of them. The effect of pulse duration on in situ acoustical estimates of fish density and their size distribution was investigated. Measurements were performed in the oligo-mesotrophic Lake Hancza (Poland) using a SIMRAD EK60 split-beam echo-sounder at 70 kHz frequency. During the survey, two similar transducers pinged alternatively through the multiplexer using 4 different pulse lengths, from short to long ones. The results show that the volume backscattering coefficient (Sv) values, equivalent of the fish biomass, were not influenced by the pulse length. However, the number of the detected fish, the mean target strength (TS), and consequently the fish density, differed significantly for the long pulse duration data. This was especially noticeable in the layer above the thermocline with dense fish populations. In this upper layer, for the long pulse the Sawada index frequently exceeded value of 0.1 leading to overestimation of the mean TS and underestimation of the fish density.