Optimization is used in different fields of engineering to solve complex problems. In image processing, multilevel thresholding requires to find the optimal configuration of thresholds to obtain accurate segmented images. In this case, the use of two-dimensional histograms is helpful because they permit us to combine information from the image preserving different features. This paper introduces a new method for multilevel image thresholding segmentation based on the improved version of the owl search algorithm (iOSA) and 2D histograms. The performance of the iOSA is enhanced with the inclusion of a new strategy in the optimization process. Moreover, in the initialization step, it is applied the opposition-based learning. Meanwhile, the 2D histograms permit to maintain more information of the image. Considering such modifications, the iOSA performs a better exploration of the search space during the early iterations, preserving the exploitation of the prominent regions using a self-adaptive variable. The iOSA is employed to allocate the optimal threshold values that segment the image by using the 2D Rényi entropy as an objective function. To test the efficiency of the iOSA, a set of experiments were performed which validate the quality of the segmentation and evaluate the optimization results efficacy. Moreover, to prove that the iOSA is a promising alternative for optimization and image processing problems, statistical tests and analyses were also conducted.