The objective of the work was to develop a non-invasive methodology for image acquisition, processing and nonlinear trajectory analysis of the collective fish response to a stochastic event. Object detection and motion estimation were performed by an optical flow algorithm in order to detect moving fish and simultaneously eliminate background, noise and artifacts. The Entropy and the Fractal Dimension (FD) of the trajectory followed by the centroids of the groups of fish were calculated using Shannon and permutation Entropy and the Katz, Higuchi and Katz-Castiglioni's FD algorithms respectively. The methodology was tested on three case groups of European sea bass (Dicentrarchus labrax), two of which were similar (C1 control and C2 tagged fish) and very different from the third (C3, tagged fish submerged in methylmercury contaminated water). The results indicate that Shannon entropy and Katz-Castiglioni were the most sensitive algorithms and proved to be promising tools for the non-invasive identification and quantification of differences in fish responses. In conclusion, we believe that this methodology has the potential to be embedded in OPEN ACCESS Entropy 2014, 16 6134 online/real time architecture for contaminant monitoring programs in the aquaculture industry.