The effective population size (Ne) is notoriously difficult to accurately estimate in wild populations as it is influenced by a number of parameters that are difficult to delineate in natural systems. The different methods that are used to estimate Ne are impacted variously by different processes at the population level, such as the life-history characteristics of the organism, gene flow, population substructure, as well as by the frequency patterns of genetic markers used and the sampling design. Here, we compare Ne estimates obtained by different genetic methods and from demographic data, and elucidate how the estimates are impacted by various factors in an exhaustively sampled and comprehensively described natural brown trout (Salmo trutta) system. In general, the methods yielded rather congruent estimates, and we ascribe that to the adequate genotyping and exhaustive sampling. Effects of violating the assumptions of the different methods were nevertheless apparent. In accordance with theoretical studies, skewed allele frequencies would underestimate temporal allele frequency changes and thereby upwardly bias Ne if not accounted for. Overlapping generations and iteroparity would also upwardly bias Ne when applied to temporal samples taken over short time spans. Gene flow from a genetically not very dissimilar source population decreases temporal allele frequency changes and thereby acts to increase estimates of Ne. Our study reiterates the importance of adequate sampling, quantification of life-history parameters and gene flow, and incorporating these data into the Ne estimation.