Diagnosis of fragmentation and landscape sustainability conditions are essential to environmental planning and sustainable management of natural resources. Land use spatial patterns and landscape structural indexes (landscape metrics, Urbanity Index-UI, and Landscape Vulnerability Index-LVI) have been proposed to assess biodiversity conservation and ecological sustainability, provided by impact of land use at Middle Mogi Guaçu watershed and its seventeen municipalities, in 2009. Land use typologies and structural indexes values were obtained based on screen digitizing of LandSat-5 imagery, for 2009. Cluster analysis and Permutational Multivariate Analysis of Variance were used to test the null hypothesis of equal degrees of fragmentation and sustainability conditions among municipalities in 2009, respectively. Land use spatial pattern showed a predominantly human occupation for watershed and its municipalities, with agricultural use as the main pressure factor. Municipalities were aggregated into three clusters related to forest fragmentation: one categorized by fifteen municipalities; the second cluster (municipality of Luiz Antônio) showing the best condition, and the third group (municipality of Araraquara) with extreme fragmentation condition. Landscape metrics related with shape, size, and core areas fragments intensify edge effects, and increase habitat isolation. The watershed showed a low naturalness and an intermediate degree of vulnerability. Ecological sustainability was different among municipalities (α = 0.05, F = 32.65 and p = 0.002), with two municipalities (Analândia and Luiz Antônio) presenting the best conditions. The most committed condition was observed, in municipalities of Rincão and Guatapará. For conservation policies to be effective must focus on the creation of ecological corridors around legally protected areas, besides creation of new legal reserves, with purpose to improve biodiversity conservation and ecological sustainability of Middle Mogi Guaçu watershed and its municipalities.